1 Introduction

Laminin-111 is a fundamental extracellular matrix (ECM) glycoprotein, mainly expressed in adult epithelial tissues. It binds to other ECM laminin isoforms and molecules and plays an important role in cell adhesion, migration, differentiation, signaling, and polarization (Streuli 1995; Hohenester and Yurchenco 2013). The γ1-chain of laminin (LAMC1) synthesizes 11 of the 15 laminin isoforms (Timpl et al. 1979; Aumailley et al. 2005) and is involved in prion disease (Machado et al. 2012).

Biologically functional interaction sequences are protein regions with significant functional roles such as molecular interactions, substrate binding, and chemical reaction catalysis. There are multiple known biologically functional interaction sequences in LAMC1 that mainly bind molecules such as integrin and nidogen and adhere to various types of cells, including cancer (Liesi et al. 1989; Mayer et al. 1993; Pöschl et al. 1994; Nomizu et al. 1997; Ponce et al. 1999; Powell et al. 2000; Kuratomi et al. 2002; Kasai et al. 2007). For instance, in LAMC1, the functional interaction sequences SLLSIINDLLEQ and TNAVGYSVYSIS highly promote neural cell growth. Also, the 58 residue-long functional interaction sequence γΙΙΙ4 (TNCPTGTTGKRCELCDDGYFGDPLGRNGPVRLCRLCQCSDNIDPNAVGNCNRLTGECL), with its essential heptapeptide NIDPNAV, only binds nidogen. Furthermore, AFSTLEGRPSAY and FDPELYRSTGHGGH bind endothelial cells and have high angiogenic activity in vivo. SETTVKYVFRLHE has high angiogenic activity in vivo, binds endothelial cells, neural cells, salivary gland cells, and three cancer cell lines (HT-1080, B16-F10, and PC12), and promotes neurite outgrowth. NDPKVLKSYYYAISDFAVGGR has high angiogenic activity in vivo, binds endothelial cells, neural cells, salivary gland cells, the cancer cell lines HT-1080, B16-F10, and NG108, and promotes the growth of the PC12 cancer cell line. RPESFAIYKRTR only binds two cancer cell lines (HT-1080 and B16-F10). Both SFSFRVDRRDTR and TSTEAYNLLLRT bind the cancer cell lines HT-1080 and B16-F10 and promote the growth of neural cells. TDIRVTLNRLNTF binds endothelial cells, salivary gland cells, and the PC12 cancer cell line.

Furthermore, a few functional interaction sequences of LAMC1 not only bind cancer cell lines but also promote their growth and metastasis. FQKLLNNLTSIKIRGTYSER binds integrin α2β1, endothelial cells, neural cells, salivary gland cells, and the cancer cell lines HT-1080, B16-F10, and NG108, and promotes the growth of the cancer cell line PC12 as well. KAFDITYVRLKF binds neural cells, promotes the growth of endothelial and salivary gland cells, binds the cancer cell lines HT-1080, B16-F10, and NG108, and promotes the growth of the cancer cell line PC12, the metastasis of the cancer cell line B16-F10, and the formation of amyloid-like fibrils in vivo. Amyloid fibrils are formed by the attachment of multiple misfolded protein aggregates in the ECM. Besides their physiological role, they compromise the healthy functioning of tissues and organs and are therefore associated with several diseases such as Alzheimer’s and Parkinson’s (Fowler et al. 2007; Eisenberg and Jucker 2012).

Phosphorylation and dephosphorylation regulate protein function by switching protein functional interaction sequences on and off (Ashcroft et al. 1999; Olsen et al. 2006). Ecto-kinases are located on the cell surface and have their catalytic site outside the cell utilizing the ATP of the ECM (Imada 1988). Ecto-kinase activity has been demonstrated in multiple cells (Ehrlich et al. 1986; Dusenbery et al. 1988; Babinska et al. 1996), while several transmembrane and ECM proteins, including laminin-111, are known to be ecto-phosphorylated (Apasov et al. 1996; Geberhiwot and Skoglund 1997; Seger et al. 1998; Zimina et al. 2007; Yalak and Vogel 2015). Protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CKII) have been reported to ecto-phosphorylate laminin-111, affecting its biological properties, such as self-assembly, and influencing its functions, like heparin binding and cell binding (Hogan et al. 1995; Kondrashin et al. 1999; Koliakos et al. 2000; Koliakos et al. 2001; Bohana-Kashtan et al. 2005; Trachana et al. 2005).

LAMC1 is part of the important laminin-111 protein and has multiple interaction sequences with different functional roles. Even though the functional interaction sequences in LAMC1 have been identified, there is little knowledge regarding their regulation by phosphorylation physiologically or in diseases. Therefore, we utilized bioinformatic tools to map the phosphorylation of LAMC1 and understand the regulation of its functional interaction sequences by specific kinases in healthy tissue in comparison with cancerous tissue. Our de novo results will provide significant aid to the scientific community as they increase the efficiency of in vitro experiments. In summary, we predicted all possible phosphorylation sites in LAMC1 using kinase binding motifs, which, in turn, led to the identification, for the first time, of all computationally identified kinases for each experimentally observed phosphorylated residue; it also predicted new putative phosphorylations. Also, we identified all point mutations found in cancer that interfere with the experimentally observed phosphorylated residues and which could interfere with the predicted phosphorylations in LAMC1. Finally, we generated an integrated phosphorylation–mutation map of LAMC1 that aims to be a useful analytic tool for the design of future targeted experiments.

2 Methods

2.1 Sequence retrieval

The sequence of LAMC1 was extracted in FASTA format (The Uniprot Consortium 2017, code P11047), which has an annotation score of 5 out of 5 and is saved as a .txt document (links in supplementary table 1).

2.2 Recording of LAMC1 functional interaction sequences

Functional interaction sequences in LAMC1 were recorded as previously (Galliou et al. 2019). An extensive literature search in PubMed (Canese and Weis 2002) was conducted to record all functional interaction sequences with a biological function, regardless of the cell type and experimental method used for identification. Overlapping functional interaction sequences were considered as one sequence. The literature-derived functional interaction sequences were matched to the consensus sequence of LAMC1 and their percentage identity was assessed by a similarity score (similarity score = number of identical residues between a literature-derived functional interaction sequence and an identified functional interaction sequence in the consensus sequence of LAMC1/length of the functional interaction sequence). A similarity score of 100% signifies two identical sequences, whereas a score of 0% signifies two completely different sequences. Functional interaction sequences in LAMC1 were saved in a .txt document (links in supplementary table 1) with a specific format (Galliou and Verrou 2019). Also, the sequence and function of recorded functional interaction sequences in LAMC1 were presented as a functional interaction sequence review (supplementary table 2).

2.3 Experimentally observed phosphorylated residues

The experimentally observed phosphorylated residues were recorded as before (Galliou et al. 2019). We scanned PhosphoSitePlus (Hornbeck et al. 2015) using the high-throughput papers (HTP) option to retrieve the phosphorylated LAMC1 residues that were experimentally assigned only through proteomic mass spectrometry. The retrieved residues were confirmed by a thorough literature search and saved in a .txt document (links in supplementary table 1) using a specific format (Galliou and Verrou 2019).

2.4 Kinase motifs

Recognition motifs of kinases indicate the sub-sequences in proteins that are required for kinase binding and phosphorylation, and were recorded as previously described (Galliou et al. 2019). A serine/threonine kinase/phosphatase motif query on Phosphomotif Finder (Amanchy et al. 2007) about all three chains of laminin-111 – LAMA1 (The Uniprot Consortium 2017, code P25391-1), LAMB1 (The Uniprot Consortium 2017, code P07942), and LAMC1 – resulted in all recognition motifs of all putative kinases that could phosphorylate LAMC1. The results were merged, sorted, and filtered in Excel to contain only kinases, and were saved in a .txt document (links in supplementary table 1) using a specific format (Galliou and Verrou 2019).

2.5 LAMC1 mutations in cancer patients

All mutations in the LAMC1 gene and protein found in cancer patients in all cancer types were retrieved from CBioPortal (Cerami et al. 2012; Gao et al. 2013), which uses Uniport for protein sequences, and saved in a .xlsx (Excel) document (links in supplementary table 1).

2.6 In silico study of LAMC1 phosphorylation

The PhosphoKin tool (Galliou and Verrou 2019) was used to study the phosphorylation of LAMC1. This tool uses as input four .txt files: the LAMC1 sequence, the functional interaction sequences, the experimentally observed phosphorylated resuidues, and the recognition motifs of kinases (links in supplementary table 1). The tool searched the recognition motifs of kinases against the LAMC1 sequence and identified the exact sub-sequences in LAMC1 that could be recognized and bound by kinases and, thus, computationally predicted all phosphorylation sites and phosphorylated residues. Also, by combining the computationally predicted phosphorylation sites in LAMC1 with the experimentally observed phosphorylated residues, it computationally identified all putative kinases for each experimentally observed phosphorylated residue in LAMC1. Then, it classified the experimentally observed and predicted phosphorylated residues into three categories: inside the functional interaction sequences, outside the functional interaction sequences, and close within six residues in proximity to functional interaction sequences.

Furthermore, we modified PhosphoKin (supplementary table 1) to also take as input an .xlsx document with the cancer mutations (supplementary table 1). Using the LAMC1 mutations file as input in PhosphoKin, we identified all missense cancer point mutations in LAMC1 that interfered with the experimentally observed and predicted phosphorylation sites of LAMC1 (supplementary table 1). Missense cancer point mutations interfering with LAMC1 phosphorylation were considered those that were located either directly on predicted phosphorylated residues or on required residues for kinase binding to phosphorylation sites according to the kinases’ recognition motifs. The missense cancer point mutations interfering with LAMC1 phosphorylation were categorized by cancer type, location relative to functional interaction sequences (inside, outside, and close within six residues proximity), and the classified mutation functional impact according to CBioPortal (tolerated and deleterious).

2.7 Mapping LAMC1 phosphorylation sites and missense point mutations in cancer

Using different styling formats we illustrated the recorded functional interaction sequences as well as the experimentally observed and predicted phosphorylated residues in the LAMC1 sequence, as previously described (Galliou et al. 2019). Also, we illustrated all missense point mutations found in all types of cancer, generating an integrated phosphorylation–mutation map of LAMC1 (supplementary figure 1), which was very useful for further analysis. Briefly, in the integrated phosphorylation–mutation map of LAMC1, the recorded functional interaction sequences were marked with boldface, the experimentally observed phosphorylated residues were marked in red-colored letters, and the predicted phosphorylation sites were highlighted in yellow. The interaction of LAMC1 residues with different kinases is described with a list of interaction rows, where each interaction row corresponds to a single kinase. The absence of interaction is displayed with ‘-’, binding of a residue with ‘X’, and phosphorylation of a residue with ‘P’. Also, the residues found mutated in cancer were underlined and their missense mutation(s) were written above them in blue color.

3 Results

3.1 Integrated phosphorylation–mutation map of LAMC1

We mapped the literature-derived, functional interaction sequences of LAMC1, the experimentally observed and predicted phosphorylated residues, and all missense point mutated residues found in different cancers (supplementary figure 1). Twenty functional interaction sequences, 22 experimentally observed phosphorylated residues (Stokes 2007; Possemato 2008; Rush 2008; Gu 2009; Ren 2009; Blasius et al. 2011; Rigbolt et al. 2011; Rikova 2011; Zhou 2011; Mertins et al. 2014, 2016), 181 predicted phosphorylated residues, and 72 missense point mutations in cancer were mapped. Of the 181 predicted phosphorylated residues, 161 were identified for the first time.

3.2 Computationally identified kinases for experimentally observed phosphorylated residues in LAMC1

Twenty-two LAMC1 phosphorylated residues have been experimentally found and 183 were predicted. All residues were sorted according to their location relative to functional interaction sequences. Of the experimentally observed phosphorylated residues, 7 were located inside functional interaction sequences, 2 were close to within six residues of functional interaction sequences, and 13 were outside functional interaction sequences (table 1). Of the predicted phosphorylated residues, 43 were located inside functional interaction sequences, 26 close to within six residues, and 141 outside (supplementary table 3). We found that 31.8% of experimentally observed phosphorylations and 23.5% of predicted phosphorylations were located inside functional interaction sequences, while 9.1% of experimentally observed phosphorylations and 14.2% of predicted phosphorylations were located close to functional interaction sequences.

Table 1 Computationally identified kinases for the experimentally observed phosphorylated residues in LAMC1
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Using the recognition motifs of kinases we computationally predicted the exact sub-sequences in LAMC1 that could be bound and phosphorylated by each kinase, and for the first time, we computationally identified all putative kinases for 20 of the 22 experimentally observed phosphorylated residues LAMC1 (table 1). Experimentally observed LAMC1 phosphorylations were inside or close to four functional interaction sequences. The experimentally observed phosphorylated residue T258 is located inside TDIRVTLNRLNTF, and the kinases PKC, CKII, CaMKII, CaMKII alpha, CaMKIV, and Chk1 were identified as its putative kinases. The fact that T258 is phosphorylated by Chk1 in vitro (Blasius et al. 2011) demonstrates the validity of our predictive approach. Furthermore, T1529 is close to SLLSIINDLLEQ, which highly promotes neurite outgrowth, with only BARK1 as a putative kinase. Inside NDPKVLKSYYYAISDFAVGGR, the residue S272 is experimentally phosphorylated with kinases PKA, CKII, and GPCRK1, and the residues Y274 and Y275 with Src as their only putative kinase. Inside RPESFAIYKRTR, the residue T166 is experimentally phosphorylated with kinases PKA, PKC, and CKII, and the residue S159 with PKA and PKC is included in its kinases. Thus, PKA and PKC that have ecto-phosphorylating activity target the functional interaction sequence RPESFAIYKRTR, which binds to fibrosarcoma HT1080 and melanoma B16F10 cancer cell lines.

3.3 Enriched kinases in the phosphorylation of LAMC1

The LAMC1 residues that were computationally identified for each kinase were counted to investigate their significance (figure 1). CKII was computationally identified for nine, while GPCR1 for six, and PKA and PKC for five experimentally observed phosphorylated residues (figure 1A). CKI and Src were identified for four experimentally observed phosphorylated residues, while the remaining kinases were for less than four residues. Moreover, CKII followed by the kinases GPCRK1, PKA, PKC, and CKI had the highest number of predicted phosphorylated residues, which were 60, 50, 46, 44, and 40 residues, respectively, (figure 1B). Therefore, five kinases (CKII, GPCRK1, PKA, PKC, CKI) were found enriched for LAMC1 phosphorylation.

Figure 1
figure 1

Number of phosphorylated residues in LAMC1 for each kinase. (A) Number of experimentally observed phosphorylated residues for each computationally identified kinase. (B) Total number of predicted phosphorylated residues by each kinase. Supplementary table 2 shows the exact phosphorylated residues for each kinase relative to functional interaction sequences.

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Furthermore, we investigated the activity of the kinases relative to the LAMC1 functional interaction sequences (figure 1). All experimentally observed phosphorylated target residues of CKI and most of CKII and GPCR1 were outside functional interaction sequences, while most target residues of PKA and PKC were inside functional interaction sequences (figure 1a). Even though the predicted phosphorylated target residues of CKII, GPCRK1, CKI, PKA, and PKC were mostly outside functional interaction sequences, PKA and PKC had a high number of predicted phosphorylated residues inside and close to functional interaction sequences (figure 1b). Therefore, of the five enriched kinases in LAMC1 phosphorylation, PKA and PKC targeted more residues inside and close to functional interaction sequences, whereas CKII, GPCRK1, and CKI targeted more residues outside functional interaction sequences.

3.4 Deleterious missense point mutations interfering with LAMC1 phosphorylation in cancer

We computationally identified all LAMC1 missense point mutations found in cancer that interfere with the experimentally observed and predicted phosphorylations in LAMC1 (supplementary table 1). In total, 96 mutations interfered with LAMC1 phosphorylation in 35 cancer types, 54 of which were deleterious and 42 were tolerated (supplementary table 4A). Most mutations were localized outside functional interaction sequences, while most mutations inside and outside functional interaction sequences were deleterious (supplementary table 4A).

The 54 deleterious and phosphorylation-interfering cancer mutations were found in 23 different cancer types (figure 2). They were mostly found in skin cancers, specifically in cutaneous melanoma, and in cancers of the female reproductive system, specifically in uterine endometrioid carcinoma. Most deleterious LAMC1 mutations in cutaneous melanoma were located inside or close to functional interaction sequences, whereas most mutations in uterine endometrioid carcinoma were localized outside LAMC1 functional interaction sequences. Eight LAMC1 mutations were found generally in lung cancer and all were outside functional interaction sequences.

Figure 2
figure 2

The number of LAMC1 deleterious point mutations and LAMC1 deleterious mutated residues that interfered with the phosphorylation of LAMC1, categorized by cancer type and location relative to functional interaction sequences in LAMC1. Data are presented as a heatmap, with different color intensities corresponding to different numbers of mutations. The red-shaded cells display the number of total mutations and the number of mutations inside, close, and outside of functional interaction sequences. Cancer-mutated residues are indicated with green-shaded cells: light green indicates that the residue was found mutated in one sample, medium-shaded green indicates that the residue was found mutated in two samples, and dark green indicates that the residue was found mutated in three samples.

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3.4.1 Cancer mutated LAMC1 residues S181, S213, R255 interfere with experimentally observed phosphorylation in LAMC1

The LAMC1 residue S181 is located outside functional interaction sequences and was found mutated to tyrosine in one sample (table 2) in lung squamous cell carcinoma (figure 2). Thus, the LAMC1 cancer mutation S181Y interferes with the residue’s experimentally observed phosphorylation by the kinases GPCRK1 and MAPKAPK2 (table 2). The LAMC1 residue S213 is outside functional interaction sequences and was found mutated to tyrosine in one sample in glioblastoma multiforme (figure 2). This residue is necessary for the experimentally observed phosphorylation of the LAMC1 residue S210 by its only kinase CKI. Therefore, cancer mutation S213Y interferes with the experimentally observed phosphorylation of residue S210 by CKI. The LAMC1 residue R255 is located inside the functional interaction sequence TDIRVTLNRLNTF and was found mutated to histidine in stomach adenocarcinoma and uterine endometrioid carcinoma. This residue is necessary for the experimentally observed phosphorylation of T258 by its putative kinases PKC, CaMKII, CaMKII-alpha, CaMKIV, and Chk1 (table 2). Thus, the enriched R255H cancer mutation interferes with the experimentally observed phosphorylation of T258 and could have a general role in cancer progression.

Table 2 Deleterious cancer point mutated LAMC1 residues interfering with the phosphorylation of experimentally observed and predicted phosphorylated residues in LAMC1
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3.4.2 Cancer-mutated LAMC1 residues R359, R657, R663, and R589 interfere with predicted phosphorylation by PKA and PKC inside functional interaction sequences

The LAMC1 residue R359 is located inside the functional interaction sequence FDPELYRSTGHGGH and it is the most enriched LAMC1 mutated residue in cancer (figure 2; table 2), as it was found mutated in six samples in total, five times to cysteine and once to histidine (data not shown). Also, it was found to be enriched in melanoma tumors, such as cutaneous melanoma (three samples), desmoplastic melanoma (one sample), melanoma (one sample), and colorectal adenocarcinoma (one sample). This residue is necessary for the predicted phosphorylation of residue T361 by PKA and PKC, and thus the enriched cancer mutation R359C,H interferes with the predicted phosphorylation of T361 by PKA and PKC inside the functional interaction sequence FDPELYRSTGHGGH.

LAMC1 residues R657 and R663 are located inside the functional interaction sequence LTPFEFQKLLNNLTSIKIRGTYSER and were both found mutated three times in cancer. The cancer-mutated residue R657 was found to have a general role in cancer as it was mutated in esophagogastric adenocarcinoma (one sample), astrocytoma (one sample), and colorectal adenocarcinoma (one sample), whereas the cancer-mutated residue R663 was found enriched in uterine endometrioid carcinoma as it was found mutated in two samples of uterine endometrioid carcinoma and one of rectal adenocarcinoma. R657 was necessary for the predicted phosphorylation of T659 and R663 for the predicted phosphorylation of S661 by PKA and PKC (table 2). Thus, the enriched cancer mutations R657C,H, and R663I,G interfere with the predicted phosphorylations of PKA and PKC inside the functional interaction sequence LTPFEFQKLLNNLTSIKIRGTYSER.

The LAMC1 residue R589 is located inside the functional interaction sequence SFSFRVDRRDTR and was found to have a general role in cancer progression as it was found mutated twice, once in uterine papillary serous carcinoma and once in prostate adenocarcinoma. This residue is required for the predicted phosphorylation of S591 by PKA and PKC, and thus the enriched cancer mutation R589H interferes with the predicted phosphorylation of S591 by PKA and PKC inside the sequence SFSFRVDRRDTR.

The LAMC1 residue T1207 is located inside the functional interaction sequence TSTEAYNLLLRT and was found to have a general role in cancer progression, as it was mutated two times, once in uterine endometroid carcinoma and once in bladder/urinary tract cancer. This residue was predicted to be phosphorylated by CKII and BARK1 and it is required for the predicted phosphorylation of T1203 by GPCRK1. Thus, the enriched cancer mutation T1207M interferes with both the predicted phosphorylation of T1207 by CKII and BARK1 inside the sequence TSTEAYNLLLRT and the predicted phosphorylation of T1203 by GPCRK1.

3.4.3 LAMC1 predicted phosphorylation interference by cancer-mutated residues outside functional interaction sequences

The LAMC1 cancer mutation P72Q was found enriched in lung squamous cell carcinoma and could interfere with the predicted phosphorylation of LAMC1 residue T68 by GPCRK1 and residue T71 by GSK-3, ERK1, ERK2, and CDK5 (figure 2; table 2). The LAMC1 cancer mutation S436Y,F was found to have a general role in cancer progression as it was mutated in colon adenocarcinoma (one sample) and small cell lung cancer (one sample), and could interfere with the residue’s predicted phosphorylation by CKII and GPCRK1. The LAMC1 cancer mutation P449L was enriched in melanoma in general, as it was mutated once in cutaneous melanoma and once in lentigo malignant melanoma, and it could interfere with the predicted phosphorylation of the LAMC1 residue S450 by DNA-PK. The LAMC1 cancer mutation E711V,K was found to have a general role in cancer progression, and the cancer mutation P713S,H was enriched in cutaneous melanoma, as the former was found mutated once in papillary renal cell carcinoma and once in hepatocellular adenoma, while the latter was found mutated in two samples of cutaneous melanoma. Both LAMC1 cancer mutations could interfere with the predicted phosphorylation of LAMC1 residue T712 by BARK1, GSK-3, ERK1, ERK2, and CDK5.

4 Discussion

Laminin-111, a crucial extracellular matrix (ECM) protein, plays a pivotal role in various cellular functions. Previous investigations by Galliou et al. (2019) and Verrou et al. (2019) delved into the phosphorylation dynamics of the α1-chain (LAMA1) and the β1-chain (LAMB1) of laminin, offering valuable insights for future research. In the current study, we aimed to extend this understanding by mapping the phosphorylation sites of LAMC1 to its functional interaction sequences and the mutations associated with cancer, to shed light on its regulatory mechanisms and potential implications for therapy.

We initially focused on identifying the functional interaction sequences within LAMC1, following a similar approach as with LAMA1 and LAMB1. It is worthwhile noting that while literature-derived functional interaction sequences were primarily based on mouse or rat sequences, their homology to human LAMC1 ensured their relevance. To bolster the confidence in our findings, we conducted a thorough assessment of sequence homology, revealing that all identified functional interaction sequences had significant similarity scores, with the majority surpassing the 90% threshold (supplementary figure 2). These sequences are compiled in our functional interaction sequences review (supplementary table 2), facilitating researchers in targeting specific regions of interest. Moreover, we acknowledge the cell-specific and potentially non-simultaneous functionality of these recorded functional interaction sequences.

Next, we employed kinase recognition motifs to computationally predict the kinases responsible for phosphorylating experimentally observed residues in LAMC1. While experimental validation is paramount, this computational approach aided in narrowing down the list of potential kinases, thus guiding the design of future experiments. Notably, we observed that most phosphorylated residues were serine and threonine residues, with a smaller fraction predicted to be tyrosine residues.

Furthermore, our analysis highlighted the enrichment of certain kinases in both experimentally observed and predicted phosphorylation events in LAMC1. Notably, three of the enriched kinases, PKA, PKC, and CKII, exhibited distinct preferences in targeting residues within or near functional interaction sequences. Also, they were predicted to target interaction sequences with cancer cell binding function, such as TDIRVTLNRLNTF, SETTVKYVFRLHE, and KAFDITYVRLKF (supplementary figure 1), suggesting involvement in LAMC1 phosphorylation in cancer. Intriguingly, PKA, PKC, and CKII have been found upregulated in several cancers (Babiker et al. 2006; Wang et al. 2007) and they possess ecto-kinase activity, suggesting a potential role for extracellular phosphorylation events in modulating LAMC1 function. These findings parallel the observations made for LAMA1 and LAMB1, indicating a shared phosphorylation mechanism across the laminin-111 molecule.

Moreover, we investigated missense point mutations in LAMC1 associated with cancer and identified phosphorylation-interfering deleterious mutated residues for the first time. Of particular interest were mutations within or close to functional interaction sequences, which could potentially disrupt essential phosphorylation events crucial for proper function, thereby contributing to disease pathogenesis. Notably, certain mutations showed enrichment in specific cancer types, suggesting their potential involvement in disease progression, while others suggested their general involvement in cancer. Furthermore, taking into consideration the location of the enriched mutations in LAMC1, our findings suggest for the first time a role of phosphorylation interference in certain LAMC1 functional interaction sequences in specific cancer types and cancer progression in general. Specifically, our findings suggest phosphorylation interference of FDPELYRSTGHGGH in melanoma and cutaneous melanoma, of FQKLLNNLTSIKIRGTYSER in uterine endometrioid carcinoma and colorectal cancer, and of TDIRVTLNRLNTF, SFSFRVDRRDTR, and TSTEAYNLLLRT generally in cancer progression.

Considering our findings from the aspect of LAMC1 structure is of great importance for unraveling future research. Certain functional interaction sequences are within a few residues in proximity in the same LAMC1 domain. For example, SFSFRVDRRDTR, SETTVKYVFRLHE, and LTPFEFQKLLNNLTSIKIRGTYSER are located in the LIVA domain, while KAFDITYVRLKF, NDPKVLKSYYYAISDFAVGGR, RPESFAIYKRTR, and TDIRVTLNRLNTF are located in the LN domain (figure 3). The LN domain is part of the E4 fragment, which is essential for the self- and co-assembly of laminin-111 and is less stable against proteolysis that degrades it by 30% (Ott et al. 1982; Colognato and Yurchenco 2000). A proteolytic cleavage in any position in the fragile-structured LVIA and LN LAMC1 domains could compromise their function. Intriguingly, a cryptic site-revealing proteolytic cleavage has been identified in the β3-chain of laminin-5 (Giannelli 1997), and protease upregulation is known to drive disease progression in cancer (Rakashanda et al. 2012). Therefore, the above functional interaction sequences may represent cryptic sites revealed by proteolysis, compromising the physiological function of LAMC1 in cancer. Given the enrichment of PKA, PKC, and CKII in LAMC1 phosphorylation physiologically and in cancer and the fact that phosphorylation can trigger conformational changes in proteins (Ritz-Gold et al. 1980), these cryptic sites could be exposed by ecto-phosphorylation.

Figure 3
figure 3

Functional interaction sequences in the structure of LAMC1, their function, and emphasis in six functional interaction sequences. The structure of LAMC1 consists of EGF-like areas, the LN and LIVA that are globular domains, and the DI & II domain that intertwines with the rest of the laminin-111 chains. Rectangles are used to depict the 20 functional interaction sequences in LAMC1. The size of each functional interaction sequence is proportional to its length. Interaction sequences are colored variously to correspond to their separate functions, according to literature. Different functions, according to literature, are displayed with distinct coloring of the rectangles; green, orange, cyan, and brown correspond to healthy cells, cancer cell lines, integrin α2β1, and nidogen binding, respectively, whereas purple corresponds to amyloid-like fiber formation. Functional interaction sequences with more than one function are multi-colored. The order of the colors is random and signifies the function of the whole functional interaction sequence. Emphasis was given to six functional interaction sequences, for which the experimentally observed and predicted phosphorylated residues and the deleterious cancer mutated residues are displayed. Red indicates an experimentally observed phosphorylation, while yellow highlights and underlined letters show a predicted phosphorylated residue and a mutated residue, respectively. The kinase(s) of phosphorylated amino acids are shown with triangles: pink triangles indicate kinases with known ecto-phosphorylation activity, whereas blue triangles indicate kinases that do not have an ecto-phosphorylation activity.

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By elucidating the interplay between phosphorylation events, protein–protein interactions, and disease-associated mutations, we lay the groundwork for future research aimed at deciphering the complex biology of laminin-111 and its role in disease pathogenesis. These findings have the potential to inform the development of novel therapeutic strategies targeting ECM proteins and associated signaling pathways, with the ultimate goal of improving patient outcomes in various diseases, including cancer. In conclusion, our comprehensive analysis provides novel insights into the phosphorylation dynamics and mutational landscape of LAMC1, highlighting its potential implications in cancer biology. These findings pave the way for further investigations into the regulatory mechanisms of laminin-111 and its role in disease with in vitro experiments, such as site-directed mutagenesis and kinase assays, and have significant implications for therapeutic interventions.